Curcumin treatment enhances bioactive metabolite accumulation and reduces enzymatic browning in soybean sprouts during storage

Highlights • Curcumin inhibits enzymatic browning in soybean sprouts during storage.• Curcumin affects the browning index, lipid damage, and antioxidant capacity.• The effects of curcumin are largely reversed by DPI, an NOX inhibitor.• The mechanism of action involves the activation of NOX-dependent H2O2 signaling.• The findings provide a new method for maintaining vegetable quality during storage.

Curcumin, a natural polyphenolic compound obtained from the rhizomes of Curcuma longa, is a highly bioactive and coloring material (EI-Hack et al., 2021;Kocaadam & Ş anlier, 2017). Curcumin has attracted much attention in food, medicine, and clinical industries (Esatbeyoglu et al., 2012;Kocaadam & Ş anlier, 2017). Moreover, curcumin is a promising therapeutic agent due to its natural antioxidant properties and strong anti-inflammatory and antimicrobial functions (Esatbeyoglu et al., 2012). However, to our knowledge, no study has evaluated the potential functions of curcumin in regulating fruit and vegetable quality during storage.
Soybean sprouts contain abundant isoflavones and polyphenols (Chon, 2013;Le et al., 2019), which are required for enzymatic browning (Tomás-Barberán & Espín, 2001). In this study, we applied curcumin, a natural polyphenol, during storage of soybean sprouts to address the following two questions. First, does curcumin aggravate or alleviate enzymatic browning and antioxidant loss in soybean sprouts during storage? Second, does curcumin regulate the accumulation of bioactive (e.g., phenolics and isoflavones) nutrients and activities of browning-related enzymes in soybean sprouts during storage, and what are the potential mechanisms? This study is the first to investigate how curcumin regulates enzymatic browning and antioxidant nutrient accumulation in a stored vegetable.

Reagent preparation
All the chemical reagents used here, including diphenyleneiodonium chloride (DPI), dimethyl sulfoxide (DMSO), and curcumin, were obtained from Macklin Biochemistry & Technique Company (Shanghai, China). For preparation of curcumin solution, 50 mg of curcumin was freshly dissolved in 1 mL of DMSO and then diluted with 100 mL of distilled water before filtering through a 0.22-μm membrane for sterilization. Ferric reducing ability of plasma (FRAP) solution was freshly prepared from 0.3 M sodium acetate buffer (pH 3.6), 0.02 M FeCl 3 , and 0.01 M 2,4,6-tripyridyl-s-triazine at a ratio of 10:1:1.

Experimental design
Soybean seeds (Glycine max L.) of Zhonghuang 37, a native cultivar provided by Shouguang Seed Co. (Weifang City, China), were used in this study. Seeds of identical size and weight were selected, surface sterilized in 1% (v/v) NaClO for 5 min, and then rinsed twice with distilled water before being steeped with distilled water at room temperature for 2 h. The seeds were then placed on trays lined with absorbent paper and kept in a dark chamber at room temperature and 90 % humidity for sprouting. The sprouts were harvested at day 5 after sowing. Before storage, the soybean sprouts were soaked with water (1% DMSO; as a control), curcumin, or curcumin + DPI (50 µM; DPI as an inhibitor of NOX was added to the curcumin solution before use) for 30 min. Subsequently, the treated soybean sprouts were manually collected, washed with distilled water, and kept at 4 • C and 70 % humidity under dark conditions. After treatment for 0, 6, 12, 18, and 24 h, the sprouts were sampled for the determination of NOX activity and H 2 O 2 content. The sprouts stored for 0, 7, and 14 d were collected, rapidly frozen, and kept at − 20 • C for subsequent analyses of weight loss, lipid damage, H 2 O 2 content, antioxidant (ascorbic acid, glutathione, and nonprotein thiol) contents, accumulation and composition of phenolics and isoflavones, total antioxidant capacity, and enzyme (SOD, CAT, PAL, PPO, and POD) activities. For all assays, three replicates of 100 seeds were included for each treatment. All concentrations used in the current study were based on our preliminary study.

Evaluation of weight loss, enzymatic browning, lipid damage, and H 2 O 2 content
Weight loss was evaluated by weighing each group of soybean samples before and after the storage period, and is presented as % weight loss compared with the initial weight. Enzymatic browning of soybean sprouts was measured at 420 nm using the method of Kim, Kim, Chung, and Moon (2014). Lipid damage was evaluated using the method of Dhindsa, Dhindsa, and Thorpe (1980) and the absorbance of thiobarbituric acid reactive substances (TBARS) was recorded at 532 and 600 nm. The H 2 O 2 content was determined spectrophotometrically at 390 nm after reaction with KI (Velikova, Yordanov, & Edreva, 2000).

HPLC analysis of phenolic and isoflavone content
Soybean samples were prepared for analysis of phenolics and isoflavones by high-performance liquid chromatography (HPLC) by the method of Kim et al. (2006). Instrumentation for HPLC analysis was as described by Kim et al. (2006).

Determination of non-enzymatic antioxidant content and total antioxidant capacity
Ascorbic acid content was determined by 2,6-dichloro-phenol-indophenol titration (Ali, Khan, Malik, & Shahid, 2016). The reduced glutathione (GSH) and non-protein thiol content was measured by monitoring the absorbance at 412 nm with the method of Nagalakshmi and Prasad (2001). Total antioxidant capacity (evaluated by Fe 3+ reducing power) was evaluated using the FRAP method (Benzie & Strain, 1996). The absorbance was recorded at 593 nm.

Enzyme activity assay
NOX activity was measured at 340 nm by the method of Grace and Logan (1996).
The activity of CAT (EC 1.11.1.6) was estimated by monitoring the decrease in the absorbance of H 2 O 2 at 240 nm (Dhindsa et al., 1980). The activity of PAL was estimated using the method of Dickerson, Pascholati, Hagerman, Butler, and Nicholson (1984). One unit of PAL activity was defined as a change of 0.01 in absorbance at 290 nm per min at 25 • C.
The activity of PPO was estimated using the method of Gawlik-Dziki et al. (2008). One unit of PPO activity was defined as a change of 0.001 in absorbance at 420 nm per min at 25 • C.
The activity of POD was estimated at 470 nm using the method of Khalil, Yusuf, Bassuony, Gamal, and Madany (2020). POD activity was expressed as mol kg − 1 min − 1 .
The activities of enzymes (NOX, SOD, CAT, PAL, PPO, and POD) were measured spectrophotometrically and expressed on a protein basis. The protein concentration of the extract was determined using the method of Bradford (1976).

Data analysis
All experiments were carried out in triplicate with a completely randomized design. All data analysis was done using Duncan's multiple range test using SPSS 20 statistical software (IBM Corp., Armonk, NY, USA). The significance level was p ≤ 0.05.

Curcumin enhances the antioxidant capacity of soybean sprouts
Compared with before storage (0 d), the total antioxidant capacity, ascorbic acid content, GSH content, and nonprotein thiol content of the soybean sprouts decreased by approximately 45 %, 75 %, 81 %, and 49

Curcumin reduces postharvest quality loss and enhances peroxide accumulation
Curcumin reduced water loss and enzymatic browning in the soybean sprouts. However, this curcumin-induced reduction in water loss and enzymatic browning was attenuated by treatment with 50 µM DPI, a specific inhibitor of NOX ( Fig. 1a and b). Treatment with curcumin decreased the water loss and browning index of soybean sprouts by approximately 52 % and 78 %, respectively, after storage for 14 d ( Fig. 1b; p < 0.05). However, the water loss and browning indexes of soybean sprouts treated with curcumin + DPI were approximately 71 % and 232 % higher, respectively, after storage for 14 d compared with those of the sprouts treated with curcumin only (Fig. 1b; p < 0.05).
Curcumin decreased lipid damage (evaluated based on the TBARS content) and H 2 O 2 content in soybean sprouts during storage ( Fig. 1c; p < 0.05). For example, curcumin treatment decreased the TBARS and H 2 O 2 content of soybean sprouts by approximately by 41 % and 27 %, respectively, after storage for 14 d (Fig. 1c). This curcumin-induced decrease in TBARS and H 2 O 2 content was reversed by DPI. For example, the TBARS and H 2 O 2 contents of sprouts treated with curcumin + DPI were approximately 24 % and 6 % higher, respectively, compared with those of sprouts treated with curcumin only after storage for 7 d ( Fig. 1c; p < 0.05).

Curcumin modulates enzyme activities
Curcumin treatment enhanced the SOD and CAT activities in stored soybean sprouts compared with the control (Fig. 3a,b). However, these curcumin-induced increases in SOD and CAT activities were reversed by the addition of 50 µM DPI (Fig. 3a,b; p < 0.05). For example, compared with the control, treatments with curcumin and curcumin + DPI increased the SOD activity of soybean sprout by approximately 71 % and 40 %, respectively, after storage for 14 d ( Fig. 3a; p < 0.05). Similarly, curcumin treatment increased the CAT activity by approximately 83 % and 244 % after storage for 7 and 14 d, respectively, compared with the control (Fig. 3b; p < 0.05). Meanwhile, compared with the CAT activities of sprouts treated with curcumin alone, those of sprouts treated with curcumin + DPI were approximately 27 % and 46 % lower after storage for 7 and 14 d, respectively ( Fig. 3b; p < 0.05).
Curcumin affected the activities of PAL, PPO, and POD in stored soybean sprouts (Fig. 3c-e), and these effects were reversed by the addition of 50 µM DPI (Fig. 3c-e). After storage for 14 d, curcumin reduced the PAL and PPO activities by approximately 74 % and 56 %, respectively, compared with the control (Fig. 3c,d; p < 0.05). After storage for 7 d, the PAL and PPO activities of the sprouts treated by curcumin + DPI were approximately 62 % and 82 % higher than those of the sprouts treated with curcumin only (Fig. 3c,d; p < 0.05). Treatments with curcumin and curcumin + DPI enhanced the POD activity of soybean sprouts stored for 14 d by approximately 64 % and 29 %, Curcumin regulates the total content of phenolics (TPC) and isoflavones (TFC), and the major components of these secondary metabolites of soybean sprouts, during storage at 4 • C. Means associated with the same letter are not significantly different (n = 3; p < 0.05). DPI, diphenyleneiodonium chloride.

Discussion
In this study, curcumin reduced the water loss, enzymatic browning, lipid damage and H 2 O 2 accumulation in stored soybean sprouts compared with controls (Fig. 1). Curcumin can simultaneously alleviate enzymatic browning and lipid damage in fruit and vegetables after harvest (Pasquariello et al., 2015). In the present study, the curcumininduced suppression of water loss, enzymatic browning, lipid damage, and H 2 O 2 content was attenuated by DPI, a specific inhibitor of NOX (Ge et al., 2015), which is a major ROS-producing enzyme (Kaur et al., 2014). This indicates that the curcumin-induced suppression of postharvest vegetable quality decline (water loss, enzymatic browning, lipid damage, and H 2 O 2 content) is closely associated with NOX-dependent H 2 O 2 production. Exogenous H 2 O 2 has been shown to drastically inhibit the browning of fresh-cut fruit (Chumyam, Faiyue, & Saengnil, 2019;Peng, Yang, Li, Jiang, & Joyce, 2008).
In the present study, curcumin enhanced NOX activity and H 2 O 2 burst in stored soybean sprouts within 24 h after treatment (Fig. 2). However, these effects of curcumin were reversed by DPI (Fig. 2). NOXmediated ROS production in Botrytis cinerea is required for curcumin to inhibit pathogen growth in the plant (Hua et al., 2019), and NOXmediated H 2 O 2 signaling has been shown to trigger the antioxidant systems of plants (Jiang & Zhang, 2003;Kaur et al., 2014). Interestingly, the pro-oxidant activity of polyphenol was found to enhance antioxidant capacity in human cells (Tedesco et al., 2021). Moreover, antioxidants such as CAT, ascorbic acid, and GSH can efficiently suppress enzymatic browning in fruit and vegetables after harvest (Qiao et al., 2021;Sikora & Ś wieca, 2018;Wu, 2014). Thus, the curcumin-induced inhibition of enzymatic browning may be attributed to its own ability to enhance antioxidant capacity, leading us to question whether curcumin improved the antioxidant capacity of stored soybean sprouts.
To test the above idea, we investigated the effects of curcumin on the activities of antioxidant enzymes (e.g., SOD and CAT) and nonenzymatic antioxidants (e.g., ascorbic acid and GSH) in soybean  sprouts. As shown in Fig. 3a,b, curcumin enhanced the SOD and CAT activities in stored soybean sprouts compared with the control. This finding is partly in accordance with Hua et al. (2019), who reported that curcumin increased the activities of antioxidant enzymes in postharvest kiwifruit. Similarly, in our study, curcumin increased the total antioxidant capacity, ascorbic acid content, GSH content, nonprotein thiol accumulation, and total phenolic and isoflavone content, in soybean sprouts compared with controls (Tables 1 and 2). How does curcumin improve antioxidant capacity? One plausible explanation may be related to the pro-oxidant activity of curcumin in the presence of transition metal ions like Fe 2+ and Cu 2+ (Yoshino et al., 2004), which may be released by senescent cells of fruit and vegetables during storage. However, the curcumin-induced enhancement in antioxidant capacity was reversed by DPI (Fig. 3a,b and Tables 1 and 2), an inhibitor of NOX. Thus, the effect of curcumin on antioxidant capacity was closely associated with NOX-dependent H 2 O 2 signaling.
PPO and POD, which are related to polyphenol degradation, along with PAL are thought to be the main enzymes responsible for enzymatic browning (Tomás-Barberán & Espín, 2001). Ascorbic acid and GSH have been shown to suppress the activity of PPO (Gacche, Warangkar, & Ghole, 2004;Landi, Degl'Innocenti, Guglielminetti, & Guidi, 2013). Thus, we investigated whether curcumin regulates the activities of these browning-related enzymes in stored soybean sprouts. As shown in Fig. 3c-e, curcumin reduced the activities of PAL and PPO but enhanced the activity of POD in soybean sprouts compared with the control. Interestingly, curcumin was found to profoundly inhibit the activities of PAL (Jhin & Hwang, 2015) and PPO (Aksoy, 2020) in vitro. This suggests that the curcumin-induced reduction in enzymatic browning can be partly attributed to the inhibition of the activities of PAL and PPO in stored soybean sprouts. However, the effects of curcumin on the activities of browning-related enzymes (PAL, PPO, and POD) were reversed by DPI (Fig. 3c-e). Thus, the PAL, PPO, and POD activities were also affected by the oxidative status, which can be regulated by curcumin in soybean sprouts during storage. Moreover, phenolics are located in vacuoles, while PPO and POD are located in plastids and other organelles (Jiang, Duan, Joyce, Zhang, & Li, 2004). This cellular compartmentalization can prevent PPO and POD from contacting phenolic substrates, effectively avoiding enzymatic browning in normal fruit and vegetables (Jiang et al., 2004). In our study, curcumin reduced lipid damage and maintained the integrity of the cellular membrane structure by increasing the ROS scavenging capacity in soybean sprouts (Table 1; Fig. 1b; Fig. 3a,b). Thus, curcumin deterred cellular decompartmentalization and prevented PPO and POD from contacting phenolics to form brown polymers (Jiang et al., 2004).
For a better understanding, we developed a hypothetical model based on NOX-mediated H 2 O 2 signaling to illustrate how curcumin hinders antioxidant loss and reduces enzymatic browning in stored soybean sprouts (Fig. 4). In this model, curcumin triggers NOX-mediated H 2 O 2 signaling, which activates the antioxidant system in stored soybean sprouts. The antioxidants inhibit the activity of PPO, a key enzyme for enzymatic browning. Moreover, the enhanced antioxidant capacity helps maintain the membrane integrity, which prevents phenolics from contacting PPO. Based on the current study and published data, this model provides a new pathway through which curcumin inhibits enzymatic browning in soybean sprouts during storage. However, additional experimental evidence is required to clarify the detailed mechanism.

Conclusions
Some interesting conclusions can be drawn from this study. First, curcumin inhibited water loss, enzymatic browning, lipid peroxidation, and H 2 O 2 content, and decreased the activities of PPO and PAL in soybean sprouts during storage. Second, curcumin triggered NOXdependent H 2 O 2 signaling to inhibit enzymatic browning. Third, curcumin enhanced the activities of antioxidant enzymes (e.g., SOD and CAT) and reduced the loss of antioxidants (e.g., ascorbic acid and GSH) in soybean sprouts during storage. This is the first study to demonstrate that curcumin suppresses enzymatic browning in stored vegetables through NOX-mediated H 2 O 2 signaling. The findings provide evidence for the application of curcumin to preserve soybean sprouts.

Declaration of Competing Interest
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Data availability
Data will be made available on request. Fig. 4. Hypothetical model for how curcumin inhibits enzymatic browning. A hypothetical model for curcumin-induced inhibition of enzymatic browning in soybean sprouts during storage via activation of NOX-mediated H 2 O 2 signaling. The sharp and blunt arrows represent positive and negative effects, respectively. Curcumin enhanced the antioxidant capacity, which inhibited the activity of PPO, and prevented contact between phenolics and PPO by maintaining the membrane integrity. For more details, see the text. AA, ascorbic acid; NOX, NADPH oxidase; PPO, polyphenol oxidase; DPI, diphenyleneiodonium chloride.